Cleavable hairpin primers

ABSTRACT

Nucleic acid constructs and methods that provide superior prevention of primer-dimers and other artifacts of false priming events are disclosed. In particular, there is disclosed a hairpin primer having a target-specific primer region, wherein the target-specific region comprises a target-binding dependent cleavage sequence; a first stem forming region 5′ of the target-specific primer region; and a second stem forming region 3′ of the target-specific primer region, wherein the second stem forming region is complementary to the first stem forming region. Methods of using the hairpin primer to amplify a target nucleic acid are also disclosed.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/037,315, filed Aug. 14, 2014, the entirety of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of genetics andmolecular biology. More particularly, it concerns the detection ofnucleic acids.

2. Description of Related Art

Nucleic acid amplification and detection techniques are frequentlyemployed in analyzing DNA samples for mutations and polymorphisms. Theyare also employed in the detection and typing of bacteria, viruses, andfungi, including those that are infectious pathogens, by analysis oftheir DNA or RNA. The most common form of nucleic acid amplification isthe polymerase chain reaction (PCR), which uses short oligonucleotidesequences called primers to initiate the reaction at a specific sitewithin the target genome. The specificity of the polymerase chainreaction is dependent upon hybridization of the primers to the targetnucleic acid. Factors affecting this specificity include the meltingtemperature (Tm), which is the temperature at which one-half of apopulation of hybridized oligonucleotides will dissociate and becomesingle stranded. Typically, a primer Tm is designed to be just above theannealing temperature set on the thermal cycling instrumentation, sothat, ideally, only those target sequences which are perfectlycomplimentary to the primer sequences will anneal, and those which arenot perfectly complimentary will not anneal. Often the temperature dropsbelow the ideal annealing temperature, such as during PCR reactions,which include reverse transcription (RT) stages, or in the case ofinstrument to instrument temperature variation. These lower than idealannealing temperatures can have adverse effects on the specificity ofthe PCR, allowing mis-priming events to unintended targets as well asprimer-primer annealing, causing unintended amplification ofprimer-dimers which can out compete the intended target amplification.

Attempts have been made previously to limit the activity of thepolymerase enzymes during the PCR set up and reverse transcription (RT)stages by using methods known as “hot-start.” These methods are intendedto reduce the formation of primer-dimers and other products ofnon-specific hybridization followed by extension. In these methods, aheat activation of polymerase is used to bring the polymerase to fullactivity. U.S. Pat. Nos. 5,773,258 5,677,152, and 5,338,671 incorporatedherein by reference use either chemical modification, or antibodybinding to reduce polymerase activity prior to heat induced cleavage ofchemical modifications or heat induced denaturation of the inhibitingantibody. These methods are beneficial in reducing the polymeraseactivity dramatically, but are known to retain varying degrees of somepolymerase activity, thereby not completely eliminating the problem. Thereverse transcriptase enzyme itself can also exhibit some DNA polymeraseactivity, which can also lead to non-specific priming events. Otherhot-start methods are known as “manual hot-start” and often includewithholding one or more reagents from the reaction until a later stage.These manual methods are often labor intensive, or require automatedliquid handling devices, and increase the risk of contamination of thePCR reaction.

Other attempts at reducing primer-dimers and other forms of non-specificamplification include the method of using linear (non-hairpin forming)primers comprising a cleavage domain within the target binding region aswell as a blocking group at the 3′ end of each primer. These methodsalso claim the use of a hotstart cleaving enzyme which is thermostable.Examples are described in US2012/0258455 and US2013/0288245, both ofwhich are incorporated herein by reference. While these methods mayprovide some increased level of specificity, they suffer from the samedrawbacks as other hot-start methods, in that some enzyme activityremains in these methods prior to heat activation.

Kaboev et. al. 2000, described using hairpin-like structured primers, toachieve an increase in specificity. This method is restrictive howeverin the practical design of PCR primer-based assays, in that thedeveloper is limited to using natural target sequences for theoligonucleotide composition of the stem.

There remains a need to provide a method of priming and amplificationthat provides superior prevention of primer-dimers and other artifactsof false priming events, particularly when the reaction is exposed tolower than ideal annealing temperatures.

SUMMARY OF THE INVENTION

Various embodiments of the present invention provide nucleic acidconstructs and methods that provide superior prevention of primer-dimersand other artifacts of false priming events.

In one embodiment, there is provided a nucleic acid constructcomprising: (a) a target-specific primer region, wherein thetarget-specific region comprises a target-binding dependent cleavagesequence; (b) a first stem forming region 5′ of the target-specificprimer region; and (c) a second stem forming region 3′ of thetarget-specific primer region, wherein the second stem forming region iscomplementary to the first stem forming region. The nucleic acidconstruct is configured such that it forms a hairpin structure undercertain conditions. As such, the nucleic acid construct is also referredto herein as a “hairpin primer.” In particular, the melt temperature(Tm) of the folded state of the hairpin primer, Tm_(hairpin), isdesigned to be lower than the Tm of the target-specific region of theprimer that is complementary to the target, Tm_(specific). Thus, as thetemperature is lowered from above Tm_(specific) to below Tm_(specific),unfolded hairpin primers anneal to target. Once annealed to the target,the target-binding dependent cleavage sequence can be recognized by acleaving agent and cleaved. Cleavage results in (1) a released fragmentcontaining the second stem forming region and a portion of thetarget-specific region that is 3′ of the cleavage site, and (2) a primerfragment. The released fragment is designed to have a significantlylower melting temperature with the target sequence than the primerfragment, and therefore will not anneal to the target once it iscleaved, allowing extension to occur from the now available 3′ end ofthe primer fragment. When the temperature is lowered below Tm_(specific)and approaches Tm_(hairpin), hairpin primers that did not anneal to thetarget, and therefore were not cleaved, fold into the hairpin state,making them unavailable for mispriming events. A person of skill in theart will be familiar with factors affecting nucleic acid hybridization,such as sequence length and G+C content, and will be able to determinethe appropriate lengths for the stem regions, target-specific regions,the released fragment, and the primer fragment in a hairpin-formingnucleic acid molecule as described herein in order to achieve theproperties mentioned above for a particular application.

The target-binding dependent cleavage sequence in the target-specificregion is a sequence that is recognized by a cleaving agent when in adouble-stranded state but is not recognized to a significant degree bythe cleaving agent when in a single-stranded state. In the hairpin stateof the nucleic acid constructs described herein, the target-specificregion is single stranded and, therefore, it is not a substrate for thecleaving agent. When the nucleic acid construct is hybridized to atarget nucleic acid, the target-binding dependent cleavage sequence isdouble stranded and, therefore, is a substrate for the cleaving agent.

There are a number of target-binding dependent cleavage sequences andcleaving agents that may be used. For example, in certain embodimentsthe target-binding dependent cleavage sequence may comprise one or moreribonucleotides and the cleaving agent may be an RNase, such as RNase H,RNase H2, RNase H2A, RNase H2B, and RNase H2C, and hotstart and/orthermophilic variants thereof. In certain aspects, the target-bindingdependent cleavage sequence contains 1, 2, 3, 4, 5, or moreribonucleotides. Typically the ribonucleotide(s) should be placed noless than 3 bases away from the second stem forming region to allow forRNase H binding and cleavage. In certain embodiments, theribonucleotide(s) are located between 3 to 12, 3 to 10, 3 to 8, 3 to 6,4 to 12, 4 to 10, 4 to 8, or 4 to 6 bases away from the second stemforming region. As a further example, the target-binding dependentcleavage sequence may comprise sequence-specific nicking enzymerecognition sequence and the cleaving agent may be a sequence-specificnicking enzyme. Sequence-specific nicking enzymes hydrolyze only onestrand of DNA, and they do so in a sequence-specific, strand-specific,and location-specific manner. Non-limiting examples of sequence-specificnicking enzymes include BbvCl and Alwl. Regardless of the target-bindingdependent cleavage sequence employed, it should be positioned such thatthe cleavage of the nucleic acid construct results in the releasedfragment having a significantly lower melting temperature with thetarget sequence than the primer fragment.

The target nucleic acid sequence may be any sequence of interest. Asample may be any sample that contains, or is suspected of containing,nucleic acids. A sample may comprise DNA (e.g., genomic DNA), RNA (e.g.,mRNA), and/or cDNA, any of which may be amplified to provide anamplified nucleic acid. In certain aspects of the invention the sampleis, for example, a subject who is being screened for the presence orabsence of one or more genetic mutations or polymorphisms. In anotheraspect of the invention the sample may be from a subject who is beingtested for the presence or absence of a pathogen. Where the sample isobtained from a subject, it may be obtained by methods known to those inthe art such as aspiration, biopsy, swabbing, venipuncture, spinal tap,fecal sample, or urine sample. In some aspects of the invention, thesample is an environmental sample such as a water, soil, or air sample.In other aspects of the invention, the sample is from a plant, bacteria,virus, fungi, protozoan, or metazoan.

As mentioned above, the first and second stem forming regions areconfigured to hybridize to each other at temperatures of Tm_(hairpin) orlower, and to be single stranded at temperatures of Tm_(specific) orabove. In certain embodiments, the first and second stem forming regionsare 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides in length,or any range therein. The stem forming regions may comprise naturallyoccurring nucleotides, non-naturally occurring nucleotides, or acombination thereof. In certain embodiments, one of the first and secondstem forming regions comprises an isoC and the other comprises an isoGat a position complementary to the isoC. In some embodiments, the firstand second stem forming regions comprise a plurality of complementaryisoC and isoG nucleotides. In certain aspects, Tm_(hairpin) is betweenabout 44° C. and 72° C., between about 45° C. and 65° C., between about55° C. and 65° C. or between about 45° C. and 55° C. The Tm_(hairpin)may be designed to be between the ideal primer annealing temperature ofthe instrument and the Tm_(specific). For example, if the annealingtemperature of the instrument was set at 58° C., the Tm_(specific) couldbe designed near 63° C. and the Tm_(hairpin) could be designed near 60°C.

In certain embodiments the stem region sequences are artificiallydesigned. Such designs can be used as a universal stem in a multiplexedassay, further reducing the bioinformatic burden of avoidingcross-reactivity. Multiple types of assays can also benefit from usingthese pre-verified universal stem sequences. These artificial stemdesigns can also be enhanced by modifications using non-natural bases(see, e.g., U.S. Pat. Nos. 5,432,272, 6,977,161, and 7,422,850 all ofwhich are incorporated herein by reference) that only bind with eachother, as well as using sequences that have been bioinformaticallyderived to not cross-react with any sequences found in nature (see,e.g., U.S. Pat. Nos. 8,624,014 and 7,226,737, both of which areincorporated herein by reference). It is typically the 3′ end of theprimer sequence that is most sensitive to non-specific amplification;therefore, avoiding risk of cross-reactivity by using artificialsequences in the stem forming regions can be advantageous.

In certain embodiments, the nucleic acid constructs disclosed hereinfurther comprise a quencher incorporated into one of the first or secondstem forming regions and a fluorophore incorporated into the other ofthe of the first or second stem forming regions. In particularembodiments, the fluorophore/quencher is incorporated at the 5′ end ofthe first stem forming region and the 3′ end of the second stem formingregion. Incorporation of a fluorophore quencher pair can be used for,for example, facilitating the simultaneous priming and detection of thetarget nucleic acid.

The nucleic acid constructs may further comprise one or more polymeraseextension blockers. In one embodiment, the nucleic acid constructcomprises an extension blocker 5′ of the target-specific primer regionand 3′ of the first stem forming region. By inserting an extensionblocker in front of (i.e., 3′ of) the first stem forming region thefirst stem forming region is not incorporated into the amplicon. Thiswould create a 5′ single-stranded region or “tag” at one end of theamplicon, if only one of a pair of nucleic acid constructs has theexntension blocker, or on both ends of the amplicon if both nucleic acidconstructs have the extension blocker. A tag could be used in a capturestep or a probing step if the stems are unique to the amplicon. In someembodiments, the nucleic acid construct comprises an extension blockerat its 3′ end. An extension blocker in this position would provide anadditional layer of protection from the nucleic acid construct primingthe synthesis of a polynucleotide prior to cleavage of the nucleic acidconstruct. The 3′ hydroxyl group may be blocked with, for example, aphosphate group, a non-naturally occurring base that is not recognizedby polymerase, or a 3′ inverted dT. Other non-limiting examples ofextension blockers that may be used in the template strand include C6-20straight chain alkylenes, iSp18 (which is an 18-atomhexa-ethyleneglycol), iMe-isodC, a hexethylene glycol monomer, syntheticnucleic acid bases, 2-O-alkyl RNA, or an oligonucleotide sequence in thereverse orientation.

The nucleic acid constructs disclosed herein are particularly useful inmultiplex PCR reactions because these constructs are configured suchthat they are closed (i.e., in a hairpin state), and thus unavailablefor mispriming or primer-dimer formation at temperatures lower than thespecific annealing temperature of the target-specific sequence to thetarget sequence, which significantly mitigates non-specificinteractions, allowing for the plex for PCR assays to be increased.Thus, various embodiments provide mixtures of a plurality of nucleicacid constructs suitable for use in multiplex PCR. Furthermore, ifdesired, the same stem forming regions may be used for all hairpinprimers in a multiplex PCR, thereby simplifying the hairpin primerdesign. For example, in one embodiment a composition is provided thatcomprises two or more populations of the nucleic acid constructsdisclosed herein, wherein (i) the target-specific primer region of onepopulation of nucleic acid constructs differs from the target-specificprimer regions of the other populations of nucleic acid constructs, and(ii) the first and second stem forming regions are the same among thetwo or more populations nucleic acid constructs.

The nucleic acid constructs can be used in PCR, reverse transcription(RT), and RT-PCR. One embodiment provides a method for amplifying atarget nucleic acid comprising: (a) contacting a sample with a firstnucleic acid construct as disclosed herein and a second nucleic acidconstruct as disclosed herein, wherein the target-specific primerregions of said first and second nucleic acid constructs are configuredas a primer pair for amplifying a target nucleic acid; (b) hybridizingthe first and second nucleic acid constructs to the target nucleic acidif present in the sample; (c) cleaving the first and second nucleic acidconstructs with a cleaving agent that recognizes the target-bindingdependent cleavage sequences of the first and second nucleic acidconstructs to obtain first and second extendable primers; and (d)extending the first and second extendable primers to amplify the targetnucleic acid.

As mentioned above, the nucleic acid constructs disclosed herein areparticularly useful in multiplexed reactions. The methods disclosedherein provide multiplexing capabilities such that a plurality of primerpairs may amplify a plurality of target nucleic acids in a single PCRreaction. In certain embodiments there are at least 6, 7, 8, 9, 10, 11,or 1° different primer pairs in a PCR reaction. In some embodimentsthere are between 8 to 100, 8 to 80, 8 to 60, 8 to 40, 8 to 20, 8 to 18,8 to 16, 8 to 12, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 10 to 20, 10to 18, 10 to 16, 10 to 12, 12 to 100, 12 to 80, 12 to 60, 12 to 40, 12to 20, 12 to 18, or 12 to 16 different primer pairs in a PCR reaction.In certain embodiments there are at least 6, 7, 8, 9, 10, 11, or 12different target nucleic acids in a PCR reaction. In some embodimentsthere are between 8 to 100, 8 to 80, 8 to 60, 8 to 40, 8 to 20, 8 to 18,8 to 16, 8 to 12, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 10 to 20, 10to 18, 10 to 16, 10 to 12, 12 to 100, 12 to 80, 12 to 60, 12 to 40, 12to 20, 12 to 18, or 12 to 16 different target nucleic acids in a PCRreaction.

Thus, one embodiment provides a multiplex method for amplifying one ormore target nucleic acids comprising: (a) contacting a sample with aplurality of nucleic acid constructs as disclosed herein, wherein thetarget-specific primer regions of said plurality of nucleic acidconstructs are configured as a plurality of primer pairs for amplifyinga plurality of target nucleic acids; (b) hybridizing the plurality oftarget nucleic acid constructs to one or more target nucleic acid(s) ifpresent in the sample; (c) cleaving the plurality of nucleic acidconstructs that have hybridized to the target nucleic acid(s) with acleaving agent that recognizes the target-binding dependent cleavagesequences of the plurality of nucleic acid constructs to obtainextendable primers; and (d) extending the extendable primers to amplifythe target nucleic acid(s). Although these methods have been describedwith both primers of a primer pair being hairpin primers disclosedherein, these methods alternatively could be performed with only oneprimer of the primer pair is a hairpin primer and the other is aconventional linear primer.

In certain embodiments, the methods further comprise detecting theamplified target nucleic acid(s). The method of detection will depend onthe manner in which the amplicons are labeled. For example, inembodiments where the nucleic acid construct comprises an extensionblocker 5′ of the target-specific primer region and 3′ of the first stemforming region, resulting in a single-stranded tag region at the 5′ ofthe amplified target nucleic acid, the detection may involve probing thetag with a complementary labeled probe or capturing the amplified targetnucleic acid by hybridization of the tag region to a complementarynucleic acid immobilized on a solid support. Probes present in the PCRreaction may comprise a blocked 3′ hydroxyl group to prevent extensionof the probes by the polymerase. The 3′ hydroxyl group may be blockedwith, for example, a phosphate group or a 3′ inverted dT. In otherembodiments, a label or labels are incorporated into the amplicon by oneor both of the primers. In certain embodiments, the amplicons may belabeled with a DNA binding dye like ethidium bromide or SYBR Green.

A labeling agent, which may also be referred to as a reporter, is amolecule that facilitates the detection of a molecule (e.g., a nucleicacid sequence) to which it is attached. Numerous reporter molecules thatmay be used to label nucleic acids are known. Direct reporter moleculesinclude fluorophores, chromophores, and radiophores. Indirect reportermolecules include biotin, which must be bound to another molecule suchas streptavidin-phycoerythrin for detection. Pairs of labels, such asfluorescence resonance energy transfer pairs or dye-quencher pairs, mayalso be employed. Labeled amplification products may be labeled directlyor indirectly. Direct labeling may be achieved by, for example, usinglabeled primers, using labeled dNTPs, using labeled nucleic acidintercalating agents, or combinations of the above. Indirect labelingmay be achieved by, for example, hybridizing a labeled probe to theamplification product.

A variety of solid supports for the immobilization of biomolecules areknown. For example, the solid support may be nitrocellulose, nylonmembrane, glass, activated quartz, activated glass, polyvinylidenedifluoride (PVDF) membrane, polystyrene substrates, polyacrylamide-basedsubstrate, other polymers, copolymers, or crosslinked polymers such aspoly(vinyl chloride), poly(methyl methacrylate), poly(dimethylsiloxane), photopolymers (which contain photoreactive species such asnitrenes, carbenes and ketyl radicals capable of forming covalent linkswith target molecules). A solid support may be in the form of, forexample, a particle (e.g., a bead or microsphere), a column, or a chip.Molecules immobilized on planar solid supports are typically identifiedby their spatial position on the support. Molecules immobilized onnon-planar solid supports, such as beads, are often identified by someform of encoding of the support. Particles may be encoded such that onesubpopulation of particles can be distinguished from anothersubpopulation. Encoding may be by a variety of techniques. For example,the particles may be fluorescently labeled with fluorescent dyes havingdifferent emission spectra and/or different signal intensities. Incertain embodiments, the particles are Luminex MicroPlex® or MagPlex®microspheres. The size of the particles in a subpopulation may also beused to distinguish one subpopulation from another. Another method ofmodifying a particles is to incorporate a magnetically responsivesubstance, such as Fe3O4, into the structure. For example, paramagneticand superparamagnetic microspheres have negligible magnetism in theabsence of a magnetic field, but application of a magnetic field inducesalignment of the magnetic domains in the microspheres, resulting inattraction of the microspheres to the field source. Combiningfluorescent dyes, particle size, and/or magnetically responsivesubstances into the particles (e.g., beads) can further increase thenumber of different subpopulations of particles that can be created.

In some embodiments, the amplified target nucleic acid(s) are detectedor otherwise analyzed by melt analysis. In certain embodiments in whichthe nucleic acid constructs lack an extension blocker 5′ of thetarget-specific primer region and 3′ of the first stem forming region,the first stem forming region can be configured to provide apredetermined increase in the melting temperature of the amplifiedtarget nucleic acid as compared to the melting temperature of theamplified target nucleic acid without the first stem forming region.Distinguishable melt points (e.g., melt points that differ by 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30° C. from one another, or any rangederivable therein) can be used to, for example, identify or distinguishbetween two or more different target nucleic acids. Non-limitingexamples of labeling agents suitable for melt analysis includeintercalating agents, double-stranded DNA binding dyes, FRET pairs, orfluor/quencher pairs. In a particular embodiment, the melt analysis usesa fluor-labeled isobase (e.g., HEX or FAM labeled isoC) on one strandand a complementary quencher-labeled isobase on the other strand (e.g.,dabcyl-labeled isoG).

In embodiments in which the nucleic acid constructs lack an extensionblocker 5′ of the target-specific primer region and 3′ of the first stemforming region, the amplification methods disclosed herein may beperformed such that initial stages are performed at a first temperatureand later stages are performed at a second temperature, which is higherthan the first. In this way, only the primers that have been copiedthrough the first stem forming region can hybridize

In another embodiment, there is provided a method of detecting a singlenucleotide polymorphism comprising: (a) contacting a sample with atleast a first and a second nucleic acid construct, each of the first andsecond nucleic acid constructs comprising: (a) a target-specific primerregion; (b) a first stem forming region 5′ of the target-specific primerregion; and (c) a second stem forming region 3′ of the target-specificprimer region, wherein the second stem forming region is complementaryto the first stem forming region; wherein the target-specific region ofthe first nucleic acid construct comprises a ribonucleotidecomplementary to a wildtype nucleotide in the target nucleic acidsequence, and the target-specific region of the second nucleic acidconstruct comprises a ribonucleotide complementary to a polymorphicnucleotide in the target nucleic acid sequence; (b) hybridizing thefirst and second nucleic acid constructs to the target nucleic acid ifpresent in the sample; (c) cleaving any of the first and second nucleicacid constructs hybridized to the target nucleic acid with RNase H tocreate an extendable primer; (d) extending the extendable primers toamplify the target nucleic acid(s); and (e) detecting the presence orabsence of the single nucleotide polymorphism by determining whether theamplified target nucleic acid(s) were amplified by one or both of thefirst and second target nucleic acid constructs. In certain aspects, themethod further comprises contacting the sample with a primer configuredto form a primer pair with both the first and second nucleic acidconstructs (i.e., the allele specific constructs). This additionalprimer may be contacted with the sample as a linear primer or as a thirdnucleic acid construct comprising: (a) a target-specific primer region,wherein the target-specific region comprises a ribonucleotide; (b) afirst stem forming region 5′ of the target-specific primer region; and(c) a second stem forming region 3′ of the target-specific primerregion, wherein the second stem forming region is complementary to thefirst stem forming region.

The methods disclosed herein may further comprise quantifying theinitial amount of the target nucleic acid(s) in the sample. Thequantification may comprise, for example, determining the relativeconcentrations of DNA present during the exponential phase of thereal-time PCR by plotting fluorescence against cycle number on alogarithmic scale. The amounts of DNA may then be determined bycomparing the results to a standard curve produced by real-time PCR ofserial dilutions of a known amount of DNA. Additionally, real-time PCRmay be combined with reverse transcription polymerase chain reaction toquantify RNAs in a sample, including low abundance RNAs.

In certain embodiments, the methods disclosed herein may furthercomprise performing a reverse transcription (RT) reaction using one ormore RT primers. Various embodiments of the present invention areparticularly useful in “one-step RT/PCR” reactions where the reversetranscription step and PCR are performed in a single closed tube withoutstepwise addition of reagents. Conventional RT-PCR methods often use thesame primers for RT and PCR. The RT and PCR steps are not typicallyperformed at the same temperature however, as many RT enzymes are heatlabile and most active at lower temperatures. Thus, the primers that arerequired to anneal at the higher annealing temperature of PCR may annealnon-specifically at the RT annealing temperature which is severaldegrees lower. The hairpin primers disclosed herein improve thespecificity at these lower temperatures by providing primers that areblocked from forming non-specific hybridizations by the hairpinstructure, in addition to comprising a specific cleavage domain.Embodiments are provided which allow two primers that are designed toanneal to the same target region to be used specifically for one-stepRT-PCR reactions. One primer is designed with a lower Tm_(specific) andis intended to act as the RT primer. The other primer is designed with ahigher Tm_(specific) and is intended to act as the PCR primer. Theprimer that has the higher Tm_(specific) is blocked from annealingnon-specifically during the RT stage by virtue of the hairpin formation.The primer with the lower Tm_(specific), may or may not have a hairpin.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is an illustration of one embodiment of a nucleic acid constructin a folded or hairpin state.

FIGS. 2A-2D illustrate embodiments in which a nucleic acid construct isin an unfolded state and not annealed to a target nucleic acid (FIG.2A), an unfolded nucleic acid construct is annealed to a target nucleicacid (FIG. 2B), the nucleic acid construct has been cleaved resulting ina released fragment and a primer fragment, and the primer fragment hasbeen extended (FIG. 2C), and nucleic acid constructs that are in thehairpin state (FIG. 2D).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Hairpin Primers

Various embodiments of the present invention provide nucleic acidconstructs that assume a folded or “hairpin” state under certainconditions. FIG. 1 illustrates one embodiment in which a nucleic acidconstruct 10 comprises a target-specific primer region 11, whichincludes a target-binding dependent cleavage sequence 12. A first stemforming region 13 is located 5′ of the target-specific primer region 11,and a second stem forming region 14 is located 3′ of the target-specificprimer region. First stem forming region 13 and second stem formingregion 14 are complementary such that they hybridize and form the stemof the nucleic acid construct when it is in a hairpin state as shown inFIG. 1. In this embodiment, nucleic acid construct 10 includespolymerase extension blocker 15. Polymerase extension blocker 15 islocated 5′ of the target-specific primer region 11 and 3′ of the firststem forming region 13. By inserting extension blocker 15 in front of(i.e., 3′ of) first stem forming region 13, first stem forming region 13is not incorporated into the amplicon. This would create a 5′single-stranded region or “tag” at one end of the amplicon, if only oneof a pair of nucleic acid constructs used in the amplification hasexntension blocker 15, or on both ends of the amplicon if both nucleicacid constructs have the extension blocker 15.

FIGS. 2A-2D illustrate an embodiment in which a nucleic acid constructis annealed to a target nucleic acid, cleaved to generate a cleavedfragment and a primer fragment, the primer fragment is extended usingthe target nucleic acid as the template. The melt temperature (Tm) ofthe folded state of nucleic acid construct 10, Tm_(hairpin), is designedto be lower than the Tm of the target-specific region of the primer thatis complementary to target nucleic acid 20, Tm_(specific). Thus, asshown in FIG. 2A, when the temperature, T, is greater thanTm_(specific), nucleic acid construct 10 is in an unfolded state and isnot annealed to target nucleic acid 20. As the temperature is loweredfrom above Tm_(specific) to below Tm_(specific), unfolded nucleic acidconstruct 10 anneals to target nucleic acid 20 as shown in FIG. 2B. Alsoas shown in FIG. 2B, stem forming regions 13 and 14 are notcomplementary to target nucleic acid sequence 20. Once annealed totarget nucleic acid sequence 20, the target-binding dependent cleavagesequence 12 is recognized by a cleaving agent and cleaved. Cleavageresults in (1) a released fragment 16 containing the second stem formingregion 14 and a portion of the target-specific region that is 3′ of thecleavage site, and (2) a primer fragment 17. Released fragment 16 isdesigned to have a significantly lower melting temperature with targetnucleic acid sequence 20 than does primer fragment 17, and willunanneal, allowing extension to occur from the now available 3′ end ofprimer fragment 17 as shown in FIG. 2C. When the temperature is loweredbelow Tm_(specific), and approaches Tm_(hairpin), nucleic acidconstructs 10 that did not anneal to the target nucleic acid sequence20, and therefore were not cleaved, fold into the hairpin state as shownin FIG. 2D, making them unavailable for mispriming events at the lowertemperatures.

As described above, a nucleic acid construct is designed to function asa “primer” under certain conditions. A “primer” is a short nucleic acid,usually a ssDNA oligonucleotide, which may be annealed to a targetpolynucleotide by complementary base-pairing. In certain embodiments,the primer has a target-specific sequence that is between 10-40, 15-30,or 18-26 nucleotides in length. The primer may then be extended alongthe target DNA or RNA strand by a polymerase enzyme, such as a DNApolymerase enzyme. Primer pairs can be used for amplification (andidentification) of a nucleic acid sequence (e.g., by the polymerasechain reaction (PCR)). In certain embodiments, the nucleic acidconstructs may contain one or more labels. Typical labels includefluorescent dyes, quenchers, radioactive isotopes, ligands,scintillation agents, chemiluminescent agents, and enzymes.

As used herein, “complementarity” describes the relationship between twosingle-stranded nucleic acid sequences that anneal by base-pairing. Forexample, 5′-AGT-3′ pairs with its complement, 3′-TCA-5′. In someembodiments, primers or probes may be designed to include mismatches atvarious positions. As used herein, a “mismatch” means a nucleotide pairthat does not include the standard Watson-Crick base pairs, ornucleotide pairs that do not preferentially form hydrogen bonds. Themismatch may include a natural nucleotide or a non-natural ornon-standard nucleotide substituted across from a particular base orbases in a target. For example, the probe or primer sequence 5′-AGT-3′has a single mismatch with the target sequence 3′-ACA-5′. The 5′ “A” ofthe probe or primer is mismatched with the 3′ “A” of the target.Similarly, the target sequence 5′-AGA-3′ has a single mismatch with theprobe or primer sequence 3′-(iC)CT-5′. Here an iso-C is substituted inplace of the natural “T.” However, the sequence 3′-(iC)CT-5′ is notmismatched with the sequence 5′-(iG)GA-3′.

An oligonucleotide that is specific for a target nucleic acid will“hybridize” to the target nucleic acid under suitable conditions. Asused herein, “hybridization,” “hybridizing,” “anneal,” or “annealing”refers to the process by which an oligonucleotide single strand annealswith a complementary strand through base pairing under definedhybridization conditions. “Specific hybridization” is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions. Permissive conditions for annealing of nucleic acidsequences are routinely determinable by one of ordinary skill in theart. Stringency of hybridization may be expressed, in part, withreference to the temperature. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Equations for calculatingT_(m), for example, nearest-neighbor parameters, and conditions fornucleic acid hybridization are known in the art.

As used herein, “nucleic acid,” “nucleotide sequence,” or “nucleic acidsequence” refer to a nucleotide, oligonucleotide, polynucleotide, or anyfragment thereof and to naturally occurring or synthetic molecules.These terms also refer to DNA or RNA of genomic or synthetic origin,which may be single-stranded or double-stranded and may represent thesense or the antisense strand, or to any DNA-like or RNA-like material.An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose. RNA may be used in the methods described hereinand/or may be converted to cDNA by reverse transcription for use in themethods described herein.

II. Cleaving Agents

The target-specific region of various embodiments of the hairpin primersdisclosed herein include a target-binding dependent cleavage sequence.The target-binding dependent cleavage sequence is a sequence that isrecognized by a cleaving agent when in a double-stranded state but isnot recognized to a significant degree by the cleaving agent when in asingle-stranded state. In the hairpin state of the nucleic acidconstructs described herein, the target-specific region is singlestranded and, therefore, it is not a substrate for the cleaving agent.When the nucleic acid construct is hybridized to a target nucleic acid,the target-binding dependent cleavage sequence is double stranded and,therefore, is a substrate for the cleaving agent.

In certain embodiments the target-binding dependent cleavage sequencemay comprise one or more ribonucleotides and the cleaving agent may bean RNase, such as RNase H, RNase H2, RNase H2A, RNase H2B, and RNaseH2C, and hotstart and/or thermophilic variants thereof RNase H and RNaseH2 are non-specific endonucleases and catalyze the cleavage of RNA in aRNA/DNA duplex, and members of the RNase H family can be found in nearlyall organisms. RNase H preferentially cleaves the 3′-O—P-bond of RNA,generating 3′ hydroxyl and 5′ phosphate products. RNase H2preferentially nicks 5′ to a ribonucleotide within the context of a DNAduplex, leaving 5′ phosphate and 3′ hydroxyl ends.

In other embodiments the target-binding dependent cleavage sequence maycomprise sequence-specific nicking enzyme recognition sequence and thecleaving agent may be a sequence-specific nicking enzyme.Sequence-specific nicking enzymes hydrolyze only one strand of DNA, andthey do so in a sequence-specific, strand-specific, andlocation-specific manner. Thus, unlike certain restriction endonucleasesthat hydrolyze both strands of duplex DNA (“cleave”), sequence-specificnicking enzymes hydrolyze only one strand of duplex DNA (“nick”). Thenicks introduced into DNA by the actions of these enzymes can serve asinitiation points for further enzymatic reactions includingpolymerization. Examples of sequence-specific nicking enzymes includeBbvCl and Alwl. Examples of sequence-specific nicking enzymes are alsodisclosed in, for example, U.S. Pat. No. 7,081,358, which isincorporated herein by reference.

III. PCR

The nucleic acid constructs disclosed herein are particularly useful insingle-plex PCR, multiplex PCR, and RT-PCR, because these constructs areconfigured such that they are closed (i.e., in a hairpin state), andthus unavailable for mispriming or primer-dimer formation attemperatures lower than the specific annealing temperature of thetarget-specific sequence to the target sequence, which significantlymitigates non-specific interactions. As used herein, “amplification” or“amplifying” refers to the production of additional copies of a nucleicacid sequence. The term “amplification reaction mixture” refers to anaqueous solution comprising the various reagents used to amplify atarget nucleic acid. These may include enzymes (e.g., a thermostablepolymerase), aqueous buffers, salts, amplification primers, targetnucleic acid, nucleoside triphosphates, and optionally, at least onelabeled probe and/or optionally, at least one agent for determining themelting temperature of an amplified target nucleic acid (e.g., afluorescent intercalating agent that exhibits a change in fluorescencein the presence of double-stranded nucleic acid).

The polymerase chain reaction (PCR) is a technique widely used inmolecular biology to amplify a piece of DNA by in vitro enzymaticreplication. Typically, PCR applications employ a heat-stable DNApolymerase, such as Taq polymerase. This DNA polymerase enzymaticallyassembles a new DNA strand from nucleotides (dNTPs) usingsingle-stranded DNA as template and DNA primers to initiate DNAsynthesis. A basic PCR reaction requires several components and reagentsincluding: a DNA template that contains the target sequence to beamplified; one or more primers, which are complementary to the DNAregions at the 5′ and 3′ ends of the target sequence; a DNA polymerase(e.g., Taq polymerase) that preferably has a temperature optimum ataround 70° C.; deoxynucleotide triphosphates (dNTPs); a buffer solutionproviding a suitable chemical environment for optimum activity andstability of the DNA polymerase; divalent cations, typically magnesiumions (Mg2⁺); and monovalent cation potassium ions. A reversetranscriptase PCR (RT-PCR) amplification procedure may be performed toreverse transcribe mRNA into cDNA. Methods of RT-PCR are well known inthe art (see Sambrook et al., 2001).

The majority of PCR methods use thermal cycling to subject the PCRsample to a defined series of temperature steps. Each cycle typicallyhas 2 or 3 discrete temperature steps. The cycling is often preceded bya single temperature step (“initiation”) at a high temperature (>90°C.), and followed by one or two temperature steps at the end for finalproduct extension (“final extension”) or brief storage (“final hold”).The temperatures used and the length of time they are applied in eachcycle depend on a variety of parameters. These include the enzyme usedfor DNA synthesis, the concentration of divalent ions and dNTPs in thereaction, and the melting temperature (Tm) of the primers. Commonly usedtemperatures for the various steps in PCR methods are: initializationstep—94-96° C.; denaturation step—94-98° C.; annealing step—50-65° C.;extension/elongation step—70-74° C.; final elongation—70-74° C.; finalhold—4-10° C.

Real-time polymerase chain reaction, also called quantitative real timepolymerase chain reaction (qPCR) or kinetic polymerase chain reaction,is used to amplify and simultaneously detect, and optionally quantify, atargeted DNA molecule. It enables both detection and quantification (asabsolute number of copies or relative amount when normalized to DNAinput or additional normalizing genes) of a specific sequence in a DNAsample. Real-time PCR may be combined with reverse transcriptionpolymerase chain reaction to quantify low abundance RNAs. Relativeconcentrations of DNA present during the exponential phase of real-timePCR are determined by plotting fluorescence against cycle number on alogarithmic scale. Amounts of DNA may then be determined by comparingthe results to a standard curve produced by real-time PCR of serialdilutions of a known amount of DNA. Various PCR and real-time PCRmethods are disclosed in U.S. Pat. Nos. 5,656,493; 5,994,056; 6,174,670;5,716,784; 6,030,787; 6,174,670, and 7,955,802, which are incorporatedherein by reference.

In qPCR the threshold cycle (Ct) reflects the cycle number at which thefluorescence generated within a reaction crosses the threshold. It isinversely correlated to the logarithm of the initial copy number. Thedetermination of the Ct value for each reaction may be related to thebaseline, background, and threshold set by software. In some qPCRmethods, a passive reference dye is used and the signal from thefluorescent reporter is divided by the signal from the reference dye toaccount for variability in the reaction medium. This calculation givesthe normalized reporter signal (Rn). The baseline refers to the initialcycles in PCR in which there is little expected change in fluorescentsignal (usually cycles 3 to 15). This baseline can be used to determinethe background for each reaction. In a multiwell reaction plate, severalbaselines from multiple wells may be used to determine the ‘baselinefluorescence’ across the plate. There are many ways to use data analysisto determine when target amplification is above the background signal(crosses the threshold). Rn can be subtracted by the background signalto give ΔRn. Other supplements to data analysis that are typicallyemployed in qPCR may be applied to the present invention. Namely, theuse of endogenous and exogenous controls, housekeeping genes, standardcurves, internal positive controls, no amplification controls, reversetranscription controls, nontreated controls, extraction controls, timepoint zeros, healthy individual controls, and negative and positivecontrols. These may be used in the present invention in order to performComparative Ct analysis (“relative quantitation”) or standard curveanalysis (“absolute quantitation”), the Pfaffl method, end-pointquantitation, qualitative results, allelic discrimination, etc.Accounting for amplification efficiency or amplification rate may beperformed by a number of methods including but not limited to: Dilutionmethod, fluorescence increase in exponential phase, Sigmoidal orlogistic curve fit, etc. The threshold may be determined by a number ofmethods including but not limited to the second derivative maximummethod, or by a multiple of standard deviations above background, etc.Endpoint quantitative analysis could be performed by a number of methodsincluding but not limited to: relative, absolute, competitive andcomparative.

Digital PCR (dPCR) involves partitioning the sample such that individualnucleic acid molecules contained in the sample are localized in manyseparate regions, such as in individual wells in microwell plates, inthe dispersed phase of an emulsion, or arrays of nucleic acid bindingsurfaces. Each partition will contain 0 or 1 molecule, providing anegative or positive reaction, respectively. Unlike conventional PCR,dPCR is not dependent on the number of amplification cycles to determinethe initial amount of the target nucleic acid in the sample.Accordingly, dPCR eliminates the reliance on exponential data toquantify target nucleic acids and provides absolute quantification.

Multiplex-PCR and multiplex real-time PCR use of multiple, unique primersets within a single PCR reaction to produce amplicons of different DNAsequences. By targeting multiple genes at once, additional informationmay be gained from a single test run that otherwise would requireseveral times the reagents and more time to perform. Annealingtemperatures for each of the primer sets should be optimized to workwithin a single reaction.

Approaches such as allele-specific PCR (AS-PCR) and allele-specificprimer extension (ASPE) detect mutations and polymorphisms usingoligonucleotide primers selected such that they selectively achieveprimer extension of either a sequence containing a variant nucleotide orthe corresponding sequence containing the wild-type nucleotide. Suchapproaches are described in, for example, U.S. Pat. Nos. 5,595,890,5,639,611, and 5,137,806, the disclosures of which are incorporated byreference. An allele-specific primer has a discriminating nucleotide.The discriminating nucleotide may be at the 3′ terminus of the primer orit may be internal (i.e., not at the 3′ terminus). In certainembodiments the internal, allele-discriminating nucleotide is at the 3′terminus of the primer fragment, in other embodiments it is at thesecond, third, fourth, or fifth nucleotide from the 3′ terminus of theprimer fragment.

IV. Isothermal Amplification

While the hairpin primers disclosed herein are useful in PCR performedon a thermal cycler over a range of temperatures, they can also benefitisothermal amplification techniques. Isothermal amplification techniquessuch as helicase-dependent amplification, nicking-enzyme amplification(NEAR) (see, e.g., WO 96/23904, incorporated herein by reference), andrecombinase polymerase amplification (RPA), allow for amplification ofthe target nucleotide(s) at a single fixed temperature. Nevertheless,variations in set annealing temperature between instruments or platformscan still be a problem, particularly in point-of-care type of devices.Assays performed on these devices can benefit from the hairpin primersdescribed herein.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. Nos. 5,137,806, 5,432,272, 5,595,890, 5,639,611,    5,656,493; 5,716,784, 5,994,056, 5,773,258 5,677,152, 5,338,671,    6,030,787, 6,174,670, 6,977,161, 7,081,358, 7,226,737, 7,955,802,    7,422,850, and 8,624,014-   U.S. Patent Publication Nos. 2012/0258455 and US2013/0288245-   International (PCT) Patent Publication No. WO 96/23904-   Kaboev et. al. Nucleic Acids Research, Vol. 28, No. 21 e94, 2000-   Sambrook et al., 2001

1. A nucleic acid construct comprising: (a) a target-specific primerregion, wherein the target-specific region comprises a target-bindingdependent cleavage sequence; (b) a first stem forming region 5 of thetarget-specific primer region; and (c) a second stem forming region 3′of the target-specific primer region, wherein the second stem formingregion is complementary to the first stem forming region.
 2. The nucleicacid construct of claim 1, wherein the target-binding dependent cleavagesequence comprises at least one ribonucleotide.
 3. The nucleic acidconstruct of claim 2, wherein there are at least 3 nucleotides betweenthe ribonucleotide and the second stern forming region.
 4. The nucleicacid construct of claim 1, wherein the target-binding dependent cleavagesequence comprises a sequence-specific cleavage domain recognized by anicking enzyme.
 5. The nucleic acid construct of claim 1, wherein thefirst and second stem forming regions are between 4 to 18 nucleotides inlength.
 6. The nucleic acid construct of claim 1, wherein one or both ofthe first and second stem forming regions comprise one or morenon-naturally occurring nucleotides.
 7. The nucleic acid construct ofclaim 6, wherein one of the first and second stem forming regionscomprises an isoC and the other comprises an isoG at a positioncomplementary to the isoC.
 8. (canceled)
 9. The nucleic acid constructof any of claim 1, wherein the Tm of the first and second stem formingregions is between 45-60° C.
 10. The nucleic acid construct of claim 1,further comprising an extension blocker 5′ of the target-specific primerregion and 3′ of the first stem forming region.
 11. The nucleic acidconstruct of claim 1, further comprising an extension blocker 3′ of thesecond stein forming region.
 12. A composition comprising two or morepopulations of nucleic acid constructs according to claim 1, wherein (i)the target-specific primer region of one population of nucleic acidconstructs differs from the target-specific primer regions of the otherpopulations of nucleic acid constructs, and (ii) the first and secondstem forming regions are the same among the two or more populations ofnucleic acid constructs.
 13. A method for amplifying a target nucleicacid comprising: (a) contacting a sample with a first nucleic acidconstruct according to claim 1 and a second nucleic acid constructaccording to claim 1, wherein the target-specific primer regions of saidfirst and second nucleic acid constructs are configured as a primer pairfor amplifying a target nucleic acid; (b) hybridizing the first andsecond nucleic acid constructs to the target nucleic acid if present inthe sample; (c) cleaving the first and second nucleic acid constructswith a cleaving agent that recognizes the target-binding dependentcleavage sequences of the first and second nucleic acid constructs toobtain first and second extendable primers; and (d) extending the firstand second extendable primers to amplify the target nucleic acid. 14.(canceled)
 15. The method of claim 13, wherein the target-bindingdependent cleavage sequences comprise a ribonucleotide and the cleavageagent is an RNase H2. 16-17. (canceled)
 18. The method of claim 13,further comprising performing a melt analysis on the amplified targetnucleic acid(s).
 19. The method of claim 18, wherein the nucleic acidconstructs lack an extension blocker 5′ of the target-specific primerregion and 3′ of the first stem forming region, and further wherein thefirst stem forming region is configured to provide a predeterminedincrease in the melting temperature of the amplified target nucleic acidas compared to the melting temperature of the amplified target nucleicacid without the first stem forming region.
 20. (canceled)
 21. Themethod of claim 13, wherein the nucleic acid constructs comprises anextension blocker 5′ of the target-specific primer region and 3′ of thefirst stem forming region, resulting in a single-stranded tag region atthe 5′ of the amplified target nucleic acid.
 22. The method of claim 21,further comprising capturing the amplified target nucleic acid byhybridization of the tag region to a complementary nucleic acidimmobilized on a solid support.
 23. A method of detecting a singlenucleotide polymorphism comprising: (a) contacting a sample with atleast a first and a second nucleic acid construct, each of the first andsecond nucleic acid constructs comprising: (a) a target-specific primerregion; (b) a first stem forming region 5′ of the target-specific primerregion; and (c) a second stem forming region 3 of the target-specificprimer region, wherein the second stem forming region is complementaryto the first stem forming region; wherein the target-specific region ofthe first nucleic acid construct comprises a ribonucleotidecomplementary to a wildtype nucleotide in the target nucleic acidsequence, and the target-specific region of the second nucleic acidconstruct comprises a ribonucleotide complementary to a polymorphicnucleotide in the target nucleic acid sequence; (b) hybridizing thefirst and second nucleic acid constructs to the target nucleic acid ifpresent in the sample; (c) cleaving any of the first and second nucleicacid constructs hybridized to the target nucleic acid with RNase H tocreate an extendable primer; (d) extending the extendable primers toamplify the target nucleic acid(s); and (e) detecting the presence orabsence of the single nucleotide polymorphism by determining whether theamplified target nucleic acid(s) were amplified by one or both of thefirst and second target nucleic acid constructs.
 24. The method of claim23, wherein the first and second nucleic acid constructions havedifferent fluorescent labels, and determining whether the amplifiedtarget nucleic acid(s) were amplified by one or both of the first andsecond target nucleic acid constructs comprises fluorescence detection.25. The method of claim 23, wherein the first and second nucleic acidconstructions have different first stem forming regions, and determiningwhether the amplified target nucleic acid(s) were amplified by one orboth of the first and second target nucleic acid constructs comprisesmelt analysis.
 26. The method of claim 23, wherein the first and secondnucleic acid constructions have different first stem forming regions andan extension blocker 3′ of the different first stem forming regions, anddetermining whether the amplified target nucleic acid(s) were amplifiedby one or both of the first and second target nucleic acid constructscomprises capturing the amplified target nucleic acid(s) on one or moresolid supports. 27-30. (canceled)